Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca2+ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (alpha(1s) or Ca(V)1.1, alpha(2), beta(1a), delta 1, and gamma), we created transgenic mice expressing a recombinant beta(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C-termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-A resolution, three-dimensional reconstruction shows a main body of 17 x 11 x 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the alpha(2)-delta 1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the beta subunit in a single docking possibility that defines the alpha 1-beta interaction. The beta subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the alpha(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.